IJA_2025v15n2

International Journal of Aquaculture, 2025, Vol.15, No.2, 45-56 http://www.aquapublisher.com/index.php/ija 50 Off-target effect is another highly concerned issue. Because the shrimp genome is huge and the information is not as comprehensive as that of model organisms, in existing studies, sequencing is mostly used to verify whether the expected sites are mutations to confirm the effect of CRISPR, but rarely comprehensively detect possible off-target sites. This brings hidden dangers of safety: Unexpected mutations caused by off-targeting may affect other gene functions and even lead to recessive negative traits. To address this problem, the conventional approach is to avoid highly homologous sequences in the genome when designing sgRNA and adopt the lowest effective concentration of Cas9/sgRNA combination. High-fidelity Cas9 variants (such as eSpCas9, SpCas9-HF1, etc.) developed in recent years have been shown to significantly reduce off-target frequency in some model animals (Matsumoto et al., 2020). In the future, these high-fidelity enzymes are also expected to be applied to shrimp gene editing experiments to improve specificity. 5 The Application Potential of CRISPR in Shrimp Disease-Resistant Breeding 5.1 Main diseases of shrimp and their genetic background There are many types of diseases in shrimp farming, including viral, bacterial and parasitic diseases, among which viral diseases are the most harmful. In addition to the widely prevalent white spot syndrome virus (WSSV), common viral diseases include taola syndrome virus (TSV), infectious subcutaneous and hematopoietic tissue necrosis virus (IHHNV), yellow head virus (YHV), etc. Among bacterial diseases, acute hepatopancreatic necrosis (AHPND, also known as EMS) is caused by Vibrio parahemolyticus carrying virulence plasmids, which has caused serious losses in Asia since its emergence around 2010 (Seibert and Pinto, 2012). The death-stealing disease (EHP) caused by microsporidium has also attracted much attention in recent years. The frequent occurrence of these diseases is often related to factors such as breeding density and environmental stress, but from the perspective of the breeding varieties themselves, there are significant differences in the susceptibility of different strains to the disease, which shows that disease resistance has a certain genetic basis. With the development of molecular biology, the immune response mechanism of shrimp to major pathogens has been gradually revealed. In the breeding program in Thailand, a line of platypal shrimp with high resistance to WSSV was selected, which is presumably possible to carry certain favorable alleles. From the perspective of immune genes, the innate immune system of shrimps includes multiple links such as pattern recognition receptors (such as β-1,3-glucan binding proteins, TLR-like receptors), signal transducers (such as MyD88, NF-κB), and effector molecules (such as antimicrobial peptides, phenol oxidases) (Sanguanrut et al., 2018). Functional mutations in any link may affect the overall anti-infection ability (Figure 1) (Lee et al., 2022). Figure 1 External white spot symptoms indicating white spot syndrome virus (WSSV) infection (Adopted from Lee et al., 2022)

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